Isolation and characterization of bacteriophage PM3 from Aeromonas hydrophila the bacterial receptor for which is the monopolar flagellum

PM3 is an Aeromonas-specific bacteriophage which was isolated and characterized on A. hydrophila strain TF7. Spontaneous mutants resistant to PM3 were non-motile having lost their characteristic monopolar flagellum. In addition, purified flagella inactivated PM3. PM3 is the first filamentous bacteriophage isolated on Aeromonas, the adsorption site for which is the monopolar flagellum.     

Campylobacter jejuni Motility Is Required for Infection of the Flagellotropic Bacteriophage F341

Previous studies have identified a specific modification of the capsular polysaccharide as receptor for phages that infect Campylobacter jejuni. Using acapsular kpsM mutants of C. jejuni strains NCTC11168 and NCTC12658, we found that bacteriophage F341 infects C. jejuni independently of the capsule. In contrast, phage F341 does not infect C. jejuni NCTC11168 mutants that either lack the flagellar filaments (ΔflaAB) or that have paralyzed, i.e., nonrotating, flagella (ΔmotA and ΔflgP). Complementing flgP confirmed that phage F341 requires rotating flagella for successful infection. Furthermore, adsorption assays demonstrated that phage F341 does not adsorb to these nonmotile C. jejuni NCTC11168 mutants. Taken together, we propose that phage F341 uses the flagellum as a receptor. Phage-host interactions were investigated using fluorescence confocal and transmission electron microscopy. These data demonstrate that F341 binds to the flagellum by perpendicular attachment with visible phage tail fibers interacting directly with the flagellum. Our data are consistent with the movement of the C. jejuni flagellum being required for F341 to travel along the filament to reach the basal body of the bacterium. The initial binding to the flagellum may cause a conformational change of the phage tail that enables DNA injection after binding to a secondary receptor.     

A Colonization Factor (Production of Lateral Flagella) of Mesophilic Aeromonas spp. Is Inactive in Aeromonas salmonicida Strains

The nine laf (lateral flagellum) genes of mesophilic aeromonads are in the Aeromonas salmonicida genome. The laf genes are functional, except for lafA (flagellin gene), which was inactivated by transposase 8 (IS3 family). A pathogenic characteristic of mesophilic aeromonads (lateral flagella) is abolished in this specialized pathogen with a narrow host range.     

Lateral flagella are required for increased cell adherence,invasion and biofilm formation by Aeromonas spp

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.     

Microbiological characteristics of Crottin goat cheese made in different seasons

General profiles and effect of season on microbiological characteristics of Crottin goat's cheese were evaluated in different batches during storage. Microorganisms determined were: mesophilic, proteolytic, halotolerant, psychotrophs, coliforms, yeasts and Staphylococcus aureus. The presence of Escherichia coli, Salmonella spp., Yersinia enterocolitica and pH and moisture content were also analyzed. E. coli, Salmonella spp. and Y. enterocolitica were not detected in the Argentinian soft goat cheese. Cheese made during winter had significant differences in mesophilic, psychrotrophics, halotolerant microorganisms and yeast between batches. In fall cheese, there were significant differences to mesophilic, proteolytic and psychrotrophics. There were not significant differences to the microbial groups in summer cheese. The variability between batches in Crottin cheese occurred during cold months, which may be attributed to the coagulation process of the cheese at environmental temperature (without any control), creating a delay in the lactic flora activity in winter. The results showed a significant influence of the seasons in the microbial population evolution during storage, suggesting that the differences in each microbial group in different seasons could be caused by differences in the predominant strains of each group. Further studies are needed for the composition of the microorganisms, where goat's products have not specific hygienic and quality standards in Argentina.     

Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.     

Discrimination Between Mesophilic and Psychrotolerant Strains in the Bacillus cereus Group Based on the PstI Digestion of the pycA Gene

A simple and rapid assay for the detection of Bacillus weihenstephanensis isolates and other psychrotolerant strains in the Bacillus cereus group was developed. It is based on the presence of a nucleotide substitution at position 795 on the housekeeping pycA gene in all B. weihenstephanensis strains. This mutation creates a PstI recognition site. It is absent in mesophilic strains in the B. cereus group. The pycA gene is amplified by PCR and the amplicons submitted to PstI digestions. In mesophilic strains, a single band of 1,718 bp in length is visualised on an agarose gel. In B. weihenstephanensis strains and in all other psychrotolerant strains from the B. cereus group, the amplicons are cleaved and two bands of 1,175 and 543 bp, respectively, are visualised. This method could be used for the screening of B. cereus collections and for the identification of psychrotolerant and mesophilic isolates from different environments.     

Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.     

Evaluation of Lactic Acid Bacteria Isolated from Fermented Plant Products for Antagonistic Activity Against Urinary Tract Pathogen <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Urinary tract infections (UTIs) are the most common infectious diseases in infants and the elderly; they are also the most common among nosocomial infections. The treatment of UTIs usually involves a short-term course of antibiotics. The purpose of this study was to identify the strains of lactic acid bacteria (LAB) that can inhibit the urinary tract pathogen Staphylococcus saprophyticus, as alternatives to antibiotics. In this study, we collected 370 LAB strains from fermented plant products and reference strains from the Bioresources Collection and Research Center (BCRC). Using spent culture supernatants (SCS), we then screened these LAB strains with for antimicrobial effects on urinary tract pathogens by the well-diffusion assay. Seven LAB strains—PM2, PM68, PM78, PM201, PM206, PM229, and RY2—exhibited inhibitory activity and were evaluated for anti-growth activity against urinary tract pathogens by the co-culture inhibition assay. Anti-adhesion and anti-invasion activities against urinary tract pathogens were evaluated using the SV-HUC-1 urothelial cell cultures. The results revealed that the survival rate of S. saprophyticus ranged from 0.9–2.96%, with the pH continuously decreasing after co-culture with LAB strains for 4 h. In the competitive adhesion assay, the exclusion and competition groups performed better than the displacement group. In the SV-HUC-1 cell invasion assay, PM201, PM206, PM229, and RY2 were found to inhibit the invasion of SV-HUC-1 cells by S. saprophyticus BCRC 10786. To conclude, RY2, PM229, and PM68 strains exhibited inhibitory activity against the urinary tract pathogen S. saprophyticus.     

Fermentation characteristics of hybrids between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae

The cryophilic wine yeasts Saccharomyces bayanus YM-84 and YM-126 were used for hybridization with the mesophilic wine yeast Saccharomyces cerevisiae OC-2. All six hybrids were stable in tetrad analysis and pulsed field gel electrophoresis, even after twenty subcultures over two years. The fermentabilities of these hybrids at a low temperature of 7°C were superior to the mesophilic wine yeast and the same as the cryophilic wine yeasts. Conversely, their fermentabilities at the intermediate temperatures of 28 and 35°C were similar to the mesophilic wine yeast. For laboratory-scale wine-making using Koshu grape juice at 10°C, the fermentability of these hybrids was superior to the mesophilic wine yeast. They also produced higher amounts of malic acid and flavor compounds such as higher alcohols, β-phenylethyl alcohol, isoamyl acetate and β-phenylethyl acetate, and lower amounts of acetic acid than those of OC-2. These results suggest that the cryophilic wine yeast S. bayanus is useful for improving the low temperature fermentation ability of wine yeast strains.     

Discrimination of Psychrotrophic and Mesophilic Strains of the Bacillus cereus Group by PCR Targeting of Major Cold Shock Protein Genes

Detection of psychrotrophic strains (those able to grow at or below 7°C) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

Molecular characterization of the genome of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production

“Natto”, regarded as a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. Natto production is disrupted by bacteriophage infection of B. subtilis (natto); thus, it is necessary to control bacteriophage infection. A bacteriophage of B. subtilis (natto), PM1, was isolated during interrupted natto production in a factory. As PM1 was shown to have a long non-contractile tail in a morphological study, it was believed to belong to the family Siphoviridae. The genome of PM1 was shown to be a linear double-stranded DNA of approximately 50 kb. Based on the results of studies using restriction endonucleases, PM1 DNA was found to be circularly permuted, similar to bacteriophage DNA without definite ends (e.g. bacteriophage T4). The nucleotide sequence of a 1.1 kb segment of PM1 was determined and used to design a PCR assay. A 0.5 kb product was amplified from eight of ten bacteriophage isolates that infect B. subtilis (natto), and the nucleotide sequences of the PCR-amplified products were identical to those of PM1, suggesting that PM1-related bacteriophages are the most prevalent infectious agents associated with the disruption of natto production. The PCR method might be useful to detect PM1-related bacteriophages and will help to control bacteriophage infection.     

First Evidence of Lysogeny in Propionibacterium freudenreichii subsp. shermanii

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Isolation and identification of new nisin-producing Lactococcus lactis subsp. lactis from milk

A method for isolating active nisin-producing strains of mesophilic lactococci was developed. Overall, 55 strains of mesophilic lactic acid bacteria were isolated from fresh cow’s milk obtained from milk farms in various regions throughout Russia; of them, 36 displayed nisin-synthesizing activity. The three most active strains were studied according to morphological, cultural, physiological, and biochemical characteristics and identified as Lactococcus lactis subsp. lactis. The species attribution of the strains studied was confirmed by the similarity of the nucleotide sequences of the 16S rRNA gene. The nucleotide sequences of the 16S rRNA genes were deposited with the GenBank under accession numbers DQ255951–DQ255954. The distinctions between these strains in physiological and biochemical characteristics and the ranges of their bactericide action on the microorganisms capable of developing in agricultural materials and food products were determined. The isolated strains displayed considerably wider ranges of action, which differed from the nisin-producing strain MGU and the commercial nisin preparation (Nisaplin), used as a biological preserving agent.     

A colonization factor (production of lateral flagella) of mesophilic Aeromonas spp. is inactive in Aeromonas salmonicida strains

The nine laf (lateral flagellum) genes of mesophilic aeromonads are in the Aeromonas salmonicida genome. The laf genes are functional, except for lafA (flagellin gene), which was inactivated by transposase 8 (IS3 family). A pathogenic characteristic of mesophilic aeromonads (lateral flagella) is abolished in this specialized pathogen with a narrow host range.     

Role of Gne and GalE in the virulence of Aeromonas hydrophila serotype O34

The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC 3.1.5.2) and Gne (EC 3.1.5.7). Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.     

Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus.

We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division. The third pleiotropic gene, pleD, is described here for the first time. A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele. Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle. Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle. No mutants defective in both flagellum biosynthesis and stalk formation were identified. Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells.     

Complete Genome Sequence of Bp7, an Escherichia coli Bacteriophage with a Wide Host Range

Chicken colibacillosis is caused by some pathogenic Escherichia coli strains. Thirty-five pathogenic antibiotic-resistant E. coli strains were used in the host range detection of bacteriophage Bp7. The phage showed a wide range of E. coli hosts (46%). The complete genome of bacteriophage Bp7 was sequenced, assembled, and analyzed. The results revealed a linear double-stranded DNA sequence of 168,066 bp harboring 791 open reading frames. The major findings from its annotation are described.